Summary

Vertebrate pigment cells are derived from neural crest cells and are a
useful system for studying neural crest-derived traits during post-embryonic
development. In zebrafish, neural crest-derived melanophores differentiate
during embryogenesis to produce stripes in the early larva. Dramatic changes
to the pigment pattern occur subsequently during the larva-to-adult
transformation, or metamorphosis. At this time, embryonic melanophores are
replaced by newly differentiating metamorphic melanophores that form the adult
stripes. Mutants with normal embryonic/early larval pigment patterns but
defective adult patterns identify factors required uniquely to establish,
maintain or recruit the latent precursors to metamorphic melanophores. We show
that one such mutant, picasso, lacks most metamorphic melanophores
and results from mutations in the ErbB gene erbb3b, which encodes an
EGFR-like receptor tyrosine kinase. To identify critical periods for ErbB
activities, we treated fish with pharmacological ErbB inhibitors and also
knocked down erbb3b by morpholino injection. These analyses reveal an
embryonic critical period for ErbB signaling in promoting later pigment
pattern metamorphosis, despite the normal patterning of embryonic/early larval
melanophores. We further demonstrate a peak requirement during neural crest
migration that correlates with early defects in neural crest pathfinding and
peripheral ganglion formation. Finally, we show that erbb3b
activities are both autonomous and non-autonomous to the metamorphic
melanophore lineage. These data identify a very early, embryonic, requirement
for erbb3b in the development of much later metamorphic melanophores,
and suggest complex modes by which ErbB signals promote adult pigment pattern
development.

The pigment pattern is another, particularly accessible, trait altered at
metamorphosis (Kelsh, 2004;
Parichy, 2006). The early
larval pigment pattern develops in the embryo from neural crest-derived
pigment cells, or chromatophores; this pattern is completed by 5 days
post-fertilization (dpf) and comprises stripes of black melanophores with
intervening yellow xanthophores. This pattern persists until metamorphosis
(∼14 dpf) when melanophores begin to differentiate outside of the early
larval stripes. During the next 2 weeks, new adult stripes begin to form as
some metamorphic melanophores migrate to sites of adult stripe formation and
other melanophores differentiate already at these sites. The result is a
juvenile pigment pattern with two `primary' stripes of melanophores, bordering
an `interstripe' of xanthophores.

Embryonic/early larval chromatophores and metamorphic chromatophores might
have commonalities as well as differences. For example, several mutants lack
chromatophore types or pigments both before and after metamorphosis
(Lister et al., 1999;
Parichy et al., 2000b;
Lamason et al., 2005). Others
exhibit defects in the adult but not in the embryo
(Parichy et al., 2000a;
Iwashita et al., 2006;
Watanabe et al., 2006).
Mutants in this latter class are interesting because they identify genes
uniquely required to establish, maintain or recruit latent precursors that
contribute to adult form. Included among these are two mutants, puma
and picasso, each having a normal early larval pigment pattern but
fewer metamorphic melanophores (Fig.
1A,B) (Parichy and Turner,
2003b; Parichy et al.,
2003; Quigley et al.,
2004). Whereas puma is required autonomously to
metamorphic melanophore precursors during pigment pattern metamorphosis, the
cellular and temporal requirements for picasso are not known.

In this study, we find that metamorphic melanophores express
erbb3b, suggesting an autonomous activity that occurs late, during
the larva-to-adult transformation. Nonetheless, we show that erbb3b
functions both autonomously and non-autonomously to the metamorphic
melanophore lineage. We also identify a major critical period for ErbB signals
during embryonic neural crest cell migration, 2 weeks before metamorphosis,
indicating a novel role for ErbB signals in establishing precursors to adult
chromatophores. Finally, we demonstrate cryptic requirements for ErbB signals
during metamorphosis itself, suggesting redundant functions with other
pathways at this later stage. Our study provides new insights into the
development of adult form and the genetic requirements of a trait expressed
before and after metamorphosis.

MATERIALS AND METHODS

Fish stocks

Fish were maintained at 26-28°C, 14L:10D
(Westerfield, 2000).
picasso mutants were recovered in screens for
N-ethyl-N-nitrosourea-induced mutations and mapped using the
partially inbred strains ABwp and wikwp.

Cell transplantation

Chimeric embryos were generated by transplanting cells at blastula stages
(3.3-3.8 hours post-fertilization) and then were reared through metamorphosis
(Parichy and Turner,
2003a).

Pharmacological Erbb inhibitor treatments

Stock solutions of AG1478 [4-(3-chloroanilino)-6,7-dimethoxyquinazoline;
Calbiochem] or PD158780
(4-[(3-bromophenyl)amino]-6-(methylamino)-pyrido[3,4-d]pyridimine; Calbiochem)
were diluted in DMSO. Fish were treated with 3 μM of either drug in 10%
Hanks solution. To facilitate penetration, 0.5% DMSO was added to all media
and embryos were dechorionated prior to treatment. Fish were reared in
agar-lined Petri dishes or glass beakers and solutions were changed daily.
Fish reared in either drug throughout development invariably died prior to
formation of the adult pigment pattern, so could not be analyzed.

Morpholino injection

A splice-blocking morpholino against erbb3b
[TGGGCTCGCAACTGGGTGGAAACAA (Lyons et al.,
2005)] was obtained from GeneTools (Eugene, OR). One- or two-cell
embryos were injected with 300-500 pg and reared through formation of the
adult pigment pattern.

For genotyping picassowp.r2e2, we amplified genomic DNA
(pcs-wpr2e2*: TTGGTTACCATTGTGGTTGTTT, TCTTCATGGTAGCTCAGA AACATC)
from individual embryos and digested PCR products with RsaI
restriction enzyme. The wild-type amplicon cuts with RsaI at position
219, whereas the mutant allele does not cut.

In situ hybridization

Analyses of mRNA distributions in embryos followed standard protocols
(Parichy et al., 2000b). In
situ hybridization on larvae followed
(Elizondo et al., 2005), but
overnight incubation was used for probes and antibodies (a detailed protocol
is available at
http://protist.biology.washington.edu/dparichy/).
For analyses of gene expression in families segregating picasso
mutant alleles, individual embryos or larvae were imaged after staining then
transferred to DNA extraction buffer and processed to determine genotypes
retrospectively.

Image analyses and statistical methods

Embryos or larvae were viewed with Olympus SZX-12 or Zeiss Lumar
stereomicroscopes, or with Zeiss Axioplan 2 or Zeiss Observer compound
microscopes. Digital images were collected with Zeiss Axiocam cameras using
Zeiss Axiovision and corrected for contrast and color balance when
necessary.

Statistical analyses were performed with JMP 7.0 (SAS Institute, Cary, NC).
For counts of melanophores, individual cells were distinguished from one
another by treating fish with epinephrine to contract melanosomes towards cell
bodies. Densities of melanophores were determined by counting melanophores
within a rectangular region delimited by: anteriorly, the anterior margin of
the dorsal fin insertion; posteriorly, the posterior margin of the anal fin
insertion; dorsally, the posterior margin of the dorsal fin insertion;
ventrally, the posterior margin of the anal fin insertion. To control for
variation in larval development stage, we tested for effects of larval size
(measured as flank height at the posterior margin of the anal fin, hpa) as a
covariate in analyses (Parichy and Turner,
2003b), and retained this factor if P<0.05, though
analyses without the co-factor yielded qualitatively equivalent results.
Analyses of melanophore densities were treated as multifactorial analyses of
variance or covariance with replicates as blocks. Residuals in all analyses
were confirmed to be normally distributed and homoscedastic. Least squares
means (correcting for size, replicate variation, or both) are presented in
figures below, with significant differences assessed by Tukey-Kramer
comparisons to preserve an experiment-wide α=0.05.

For analyses of embryonic critical periods for Erbb signals in kit
mutant larvae (see below), adult pigment patterns were scored qualitatively
for stripe disruption. Breaks in stripes were considered present when three or
fewer melanophores were present over a defined anterior-posterior length, as
scaled by hpa (above): stripes exhibiting breaks ≤0.5 hpa were scored `0';
breaks between 0.5 and 1 hpa were scored `1'; breaks >1 hpa were scored
`2'. Dorsal and ventral stripes were scored individually, then summed to
generate a `stripe break score' of 0-4. To test for differences among
treatment groups, we compared ordinal scores using non-parametric Wilcoxon
tests and contingency table analyses. Both methods yielded equivalent results;
for simplicity, we present only the former (complete analyses available on
request).

RESULTS

Metamorphic melanophore development requires erbb3b

To learn when picasso mutants first exhibit pigment pattern
defects, we examined embryos and early larvae, and we imaged individual fish
daily from early larval stages through formation of the adult pigment pattern.
Pigment cell complements of embryos and early larvae were normal
(Fig. 1C,D). Subsequently,
picasso mutants largely failed to develop metamorphic melanophores,
particularly in the mid-trunk, and instead retained early larval melanophores
even as adults (Fig. 1E-L). In
the posterior trunk of picasso mutants, seemingly more complete
melanophore stripes form (Fig.
1B). Posterior stripes in picasso mutants appear more
complete because more embryonic/early larval melanophores become situated in
the position of adult stripes in this region, and because more metamorphic
melanophores differentiate near these cells when compared with the mid-trunk
(Fig. 2).

We mapped picasso to chromosome 23 in the vicinity of
erbb3b and found that picasso failed to complement an excess
neuromast phenotype of an erbb3b-null allele (all of these alleles
are recessive and homozygous viable, though weaker than wild type)
(Lyons et al., 2005).
erbb3b cDNAs had premature stop codons in each of two
picasso alleles (Fig.
3), demonstrating that the picasso phenotype arises from
mutations in erbb3b.

In embryos, erbb3b is expressed in neural crest cells and glia
(Lyons et al., 2005). In
metamorphosing larvae, we similarly found erbb3b expression in glia
(Fig. 4A). To determine whether
erbb3b might be expressed in other tissues below the threshold of
detection by in situ hybridization, we used RT-PCR. We detected
erbb3b transcripts in both isolated melanophores and in juvenile fin
(comprising melanophores, melanophore precursors, bone, skin, vasculature and
other cell types; Fig. 4C). We
also detected the erbb3b paralogue, erbb3a, in glia and fin,
though not in metamorphic melanophores
(Fig. 4B,C). As ErbB receptors
act as heterodimers, we tested where other ErbB genes are expressed
(Fig. 4C): erbb2 was
expressed in metamorphic melanophores and in fin; egfr was not
expressed in metamorphic melanophores, although it was expressed in fin; and
we could not detect erbb4 in melanophores or in fin (data not shown).
erbb2 and egfr are also widely expressed in zebrafish
embryos (Goishi et al., 2003;
Lyons et al., 2005).

To determine what steps in metamorphic melanophore development require
erbb3b, we examined molecular markers
(Fig. 5A-F). picasso
mutants were deficient during metamorphosis for cells expressing early neural
crest markers (crestin, sox10), as well as early and late markers of
the melanophore lineage (mitfa, dct). picasso mutants also
had transiently fewer cells expressing xanthophore lineage markers (xdh,
csf1r) and fewer myelin basic protein+
(mbp+) glia (Fig.
5G,H and data not shown).

erbb3b functions autonomously and non-autonomously to the
metamorphic melanophore lineage

erbb3b might promote adult pigment pattern formation by acting
autonomously to the metamorphic melanophore lineage, but also could have
non-autonomous effects if, for example, erbb3b-dependent cells
provide signals required by metamorphic melanophores or their precursors. To
test these possibilities, we constructed genetic mosaics by transplanting
cells between blastula stage embryos.

If erbb3b acts autonomously to the metamorphic melanophore
lineage, then wild-type melanophores should develop in picasso
mutants and these cells should form wild-type stripes. If erbb3b acts
non-autonomously, then wild-type melanophores should develop where
picasso mutant melanophores develop (anteriorly and posteriorly), but
not where picasso mutant melanophores are absent (mid-trunk)
(Fig. 1B). Wild-type
(β-actin::EGFP+) → picasso chimeras developed
wild-type metamorphic melanophores at high density anteriorly and posteriorly
(Fig. 6A,B), but often
developed few if any metamorphic melanophores in the mid-trunk
(Fig. 6A,C), like
picasso mutants. In reciprocal picasso
(β-actin::EGFP+) → wild-type chimeras, we never found
donor picasso mutant metamorphic melanophores in the adult pigment
pattern. These findings suggest both non-autonomous and autonomous roles for
erbb3b.

Given that metamorphic melanophores express erbb3b and
erbb2, differences between wild-type and picasso mutant
melanophores could be further revealed as differences in their abilities to
populate a flank lacking melanophores. We therefore transplanted wild-type or
picasso mutant cells to nacrew2 mutant hosts,
which lack their own melanophores because of a mutation in mitfa,
which functions cell-autonomously in melanophore specification
(Lister et al., 1999;
Parichy and Turner, 2003a). In
wild-type → nacre chimeras, embryonic/early larval melanophores
often developed, and metamorphic melanophores differentiated to form patches
of stripes (Fig. 6D). In
picasso → nacre chimeras, embryonic/early larval
melanophores developed about as often, but metamorphic melanophores did not
appear and, instead, embryonic/early larval melanophores persisted into the
adult (Fig. 6E). Interestingly,
metamorphic melanophores failed to develop in picasso →
nacre chimeras, even anteriorly and posteriorly; this difference from
picasso mutants may arise because the melanophore-free nacre
background would preclude community effects from contributing to pattern
regulation in these regions (Fig.
2) (Parichy et al.,
2000b; Parichy and Turner,
2003b). Together, genetic mosaic analyses indicate that ErbB
signals are required autonomously and non-autonomously during metamorphic
melanophore development.

ErbB activity is required in the embryo for metamorphic melanophore
development

The adult pigment pattern of picasso mutants could reflect
erbb3b activities early or late. For example, erbb3b could
function in the embryo to establish a population of precursors that
differentiates at metamorphosis. Or erbb3b could act later in
maintaining or expanding such a population, or still later, during their
differentiation into metamorphic melanophores. To distinguish among these
possibilities, we blocked ErbB signaling using pharmacological inhibitors
AG1478 (Levitzki and Gazit,
1995; Lyons et al.,
2005; Levitzki and Mishani,
2006) and PD158780 (Fry et
al., 1997; Rewcastle et al.,
1998; Frohnert et al.,
2003). Preliminary analyses showed that treating wild-type embryos
with either AG1478 or PD158780 resulted in excess neuromasts that phenocopy
erbb3b mutants (data not shown)
(Lyons et al., 2005). As both
drugs inhibit kinase activity by interfering with ATP-binding sites, and
wild-type Erbb3 already has impaired or absent kinase activity
(Guy et al., 1994), inhibitors
presumably suppress signals originating with erbb3b:erbb2, erbb3:egfr or other
heterodimers. Functions of these receptors that are independent of kinase
activity should not be affected.

Wild-type embryos treated with AG1478 developed normal early larval pigment
patterns. When these same fish metamorphosed, however, their pigment patterns
and melanophore densities were indistinguishable from picasso mutants
(Fig. 7B,E). By contrast, fish
treated with AG1478 during the pre-metamorphic (early larval) period or during
metamorphosis, developed adult pigment patterns and melanophore densities
indistinguishable from controls (Fig.
7A,C-E; but see below). Treatment of wild-type fish with PD158780
yielded identical results (Fig.
8 and data not shown).

To test whether the embryonic requirement for ErbB signaling is unique to
zebrafish, we examined two more species. We chose D. albolineatus
because its more uniform pigment pattern
(Fig. 7F) might depend on
mechanisms different than zebrafish
(Quigley et al., 2005;
Mills et al., 2007). Danio
albolineatus embryos developed defects in metamorphic melanophores
similar to D. rerio when treated with AG1478
(Fig. 7G) or PD158780 (data not
shown). We also examined D. nigrofasciatus
(Fig. 7H), in which few
metamorphic melanophores develop and, instead, most embryonic/early larval
melanophores persist and reorganize to form adult stripes
(Quigley et al., 2004). If
AG1478 effects are limited to metamorphic melanophores, then the D.
nigrofasciatus pigment pattern should be refractory to perturbation.
Consistent with this prediction, D. nigrofasciatus embryos treated
with AG1478 developed adult pigment pattern defects
(Fig. 7I) less severe than
those of zebrafish or D. albolineatus.

Autonomous and non-autonomous roles for erbb3b in pigment
pattern metamorphosis. (A) Wild-type → picasso
chimeras frequently developed wild-type melanophores in stripes at high
density anteriorly (arrows, left) but at lower density in the mid-trunk (small
arrows, right; 75% of chimeras developed donor melanophores; chimeras with
donor cells and total reared: n=24, 64, respectively). A wild-type
midbody lateral line is misrouted as well. (B) Melanophores at high
density anteriorly that are either donor-derived (EGFP+) or
host-derived (EGFP-). (C) Melanophores in the mid-trunk are
more spread, which is typical at low density. In reciprocal picasso→
wild-type chimeras, we did not observe donor metamorphic melanophores
(n=7, 50). (D) Wild-type → nacre chimeras
developed patches of donor-derived metamorphic melanophores that populated
stripes (arrow) and scales (84% of chimeras developed metamorphic
melanophores; n=75, 155). Persisting embryonic/early larval
melanophores (arrowheads) are identifiable by location, large size and browner
color (Quigley et al., 2004).
(E) picasso → nacre chimeras developed
melanophores (arrowheads), but did not develop metamorphic melanophores [79%
of chimeras developed embryonic/early larval melanophores or fin melanophores
(not shown); n=58, 195]. Donor cells in all chimera combinations
contributed at similar frequencies to other derivatives, including muscle,
epidermis, eye and neurons of the lateral line. Scale bars: in A, 500 μm;
in B, 200 μm for B,C; in D, 1 mm for D,E.

In mammalian systems, AG1478 is highly selective for EGFR-dependent signals
and is less effective against other ErbB receptors
(Levitzki and Gazit, 1995),
whereas PD158780 is highly effective against all ErbB family members
(Fry et al., 1997;
Frohnert et al., 2003;
Stonecypher et al., 2005).
Specificities in zebrafish are not known. Although the similarity of adult
pigment patterns between drug-treated fish and the picasso mutant is
consistent with the suppression of erbb3b-dependent signals, we would expect
these inhibitors to affect signaling through other ErbB receptors as well,
particularly as protein tyrosine kinase domains are highly conserved between
zebrafish and human orthologues (e.g. domain-specific identities,
similarities: Erbb2, 84%, 92%; Egfr, 87%, 95%; Erbb3, 75%, 87%). We therefore
repeated these experiments on picasso mutants: if signals independent
of erbb3b are inhibited, then the severity of the picasso
phenotype should be enhanced. When we treated sibling
picassowp.r2e2 and picassowp.r2e2/+
embryos with AG1478 for the first 4 days of development, homozygotes
unexpectedly developed edema and died by 7 dpf. Thus, a single copy of
erbb3b protects against AG1478-dependent lethality, implying that
erbb3b has functions that are independent of kinase activity (that,
itself, presumably originates with erbb3b:erbb2 or erbb3b:egfr heterodimers).
Consistent with this idea are several studies that have revealed
kinase-independent activities of receptor tyrosine kinases, including Erbb3
(Offterdinger et al., 2002;
Rawls and Johnson, 2003;
Massie and Mills, 2006;
Hsu and Hung, 2007).

These data indicate that ErbB signals are required in embryos for adult
pigment pattern formation. To further test this conclusion, we sought an
independent means of blocking erbb3b activity. We reasoned that the
limited perdurance of morpholino oligonucleotides (3-5 days) should allow us
to knock-down erbb3b in the embryo, while permitting later activity
at metamorphosis (Nasevicius and Ekker,
2000; Mellgren and Johnson,
2004). We therefore injected embryos with a morpholino
oligonucleotide against erbb3b
(Lyons et al., 2005) and
raised them into adults. Morpholino-injected fish showed defects qualitatively
similar to picasso mutants (Fig.
7K).

Overall then, two independent lines of evidence show that erbb3b
is required early for much later adult pigment pattern development.
Specifically, the erbb3b mutant adult pigment pattern phenotype can
be phenocopied in wild-type fish by: (1) embryonic knockdown of
erbb3b via morpholino injection; and (2) treating embryos with either
of two pharmacological inhibitors that, in this context, are specific to
erbb3b-dependent signals.

We treated kit mutant embryos beginning between 8 hpf and 70 hpf
for periods of 2-26 hours. Such analyses across multiple independent
experiments revealed peak sensitivities for adult pigment pattern formation
between ∼14 and 22 hpf, with affected individuals developing stripe
defects reminiscent of picasso mutants (AG1478:
Fig. 10A-C,E,F; PD158780: data
not shown). As adult pigment patterns were comparatively refractory to
treatments after ∼22 hpf, we asked whether longer treatments at later
stages would enhance adult pattern defects. Treating embryos between 26 and 48
hpf did not alter later phenotypes (Fig.
10F), consistent with an earlier critical period. Finally, because
erbb3b, erbb2 and egfr are expressed as early as 8-11 hpf
[(Goishi et al., 2003;
Thisse and Thisse, 2004;
Lyons et al., 2005); data not
shown], we tested whether ErbB signals have reiterated activities by treating
embryos twice. When early treatments (8-11 hpf) were combined with later
treatments (beginning at 22 hpf and later), we observed more severe
melanophore deficiencies in the adult. Remarkably, treatments at least 8 hours
apart often resulted in spatially separated melanophore-deficient patches
(e.g. Fig. 10D). The increased
severity of these defects suggests early and late functions even in the
embryo: defects arising from early ErbB kinase inhibition can presumably be
regulated so long as ErbB function is allowed later.

The major critical period for ErbB signals (∼14-22 hpf) corresponds
approximately to the time when neural crest cells are migrating at the axial
levels affected in the picasso mutant
(Raible et al., 1992;
Vaglia and Hall, 2000).
Therefore, we explored the role of erbb3b in the early patterning of
neural crest-derived cells. In comparison with wild-type siblings,
picasso mutants at 26 hpf had similar numbers of cells expressing the
pan-neural crest marker crestin, but these cells did not localize at
sites of ganglion formation in the medial migratory pathway and were instead
found further ventrally (Fig.
11A). We observed a similar defect for cells expressing
mitfa (Fig. 11B). By
contrast, we did not find clear defects in the distributions of
dct+ melanoblasts
(Fig. 11C) or cells in the
lateral migratory pathway, consistent with the normal patterning of
picasso mutant embryonic/early larval melanophores. Finally, given
the defects in ventromedial migrating cells, and defects in the peripheral
nervous system of mammalian ErbB3 and ErbB2 mutants
(Britsch et al., 1998;
Britsch, 2007), we examined
ganglion development. We observed gross reductions in the numbers of dorsal
root and sympathetic ganglia in picasso mutant larvae at 12 dpf
(Fig. 11D). These data reveal
an erbb3b-dependence of neural crest morphogenesis that correlates
with the early erbb3b-dependence of adult pigment pattern
formation.

The preceding experiments demonstrated a critical period for ErbB signaling
in embryos. During later development, metamorphic melanophores express both
erbb3b and erbb2 (Fig.
4C), but inhibitor treatments during metamorphosis had no effect
on the adult pigment pattern (Fig.
7C-E). This suggests redundancies with other pathways, regulation
in cell behaviors, poor penetration into tissues or higher thresholds for
inhibition. Given these possibilities, we sought to further test roles for
ErbB signals during metamorphosis. As higher doses were lethal, we re-tested
the inhibitors on sensitized mutant backgrounds: kit, csf1r
j4e1 and kit/+ csf1r/+. We chose these
because they reveal distinct populations of metamorphic melanophores
(Johnson et al., 1995;
Parichy et al., 1999;
Parichy et al., 2000b):
kit mutants lack early metamorphic melanophores, but retain late
metamorphic melanophores; csf1r mutants retain early metamorphic
melanophores, but are missing late metamorphic melanophores. Comparing further
deficits should therefore indicate whether one or the other population has a
greater requirement for ErbB signaling. Finally, we also examined D.
albolineatus because of the differences in melanophore development in
this species compared with zebrafish.

In each background, we observed moderate to severe reductions in
metamorphic melanophore numbers upon treatment with AG1478 or PD158780 during
metamorphosis (Fig. 12; 19-58%
fewer melanophores than corresponding controls), changes that were
considerably more severe than observed for wild-type larvae
(Fig. 7D,E; 3% fewer
melanophores than controls). Furthermore, kit mutant and
csf1r mutant zebrafish exhibited similar reductions in residual
melanophore numbers, suggesting that ErbB signals are required by precursors
to both early and late metamorphic melanophores. Consistent with the
specificity of these effects, the picassowp.r2e2 mutant
pigment pattern defect was not enhanced under these conditions and
kit mutants treated with AG1478 exhibited pigment patterns that fell
within the range observed for fish doubly mutant for kit and
picassowp.r2e2 (data not shown). These data support a
model in which ErbB signals are essential in the embryo but also function
redundantly with other pathways during metamorphosis.

This study indicates that adult pigment pattern formation requires ErbB
signaling in the embryo. Our analyses used pharmacological inhibitors that
several lines evidence suggest are specific to ErbB-dependent signals. First,
we observed the same phenotypes with two inhibitors. Second, pigment pattern
defects phenocopied erbb3b-null alleles. Third, inhibitors failed to
enhance defects of these null alleles. Fourth, similar defects resulted from
morpholino-knockdown of erbb3b. Our results thus point to a model in
which ErbB signals - depending in part on erbb3b - play an essential embryonic
role in promoting much later adult pigment pattern formation.

This early critical period contrasts with other genes. For example,
csf1r and puma are required during pigment pattern
metamorphosis (Parichy and Turner,
2003a; Parichy et al.,
2003) and kit is required during pattern formation in the
fin (Rawls and Johnson, 2001).
The critical period for ErbB signals in zebrafish also may be earlier than for
Ednrb of mouse (Shin et al.,
1999). Nevertheless, our analyses can suggest only a range of
times: both drugs act rapidly and are quickly reversible
(Fry et al., 1997;
Lenferink et al., 2001;
Levitzki and Mishani, 2006),
but we do not know how long it takes for concentration changes in solution to
reach beneath the epidermis. The peak sensitivity observed for embryos treated
between 14-22 hpf may therefore indicate somewhat later critical periods,
presumably during neural crest migration.

We can envisage at least two complementary models in which embryonic ErbB
signals contribute to later metamorphic melanophore development. In the first
model, these signals act autonomously to establish precursors of metamorphic
melanophores. This activity could be specific to metamorphic melanophores or
could apply to a broader range of neural crest derivatives. For example, both
pigment cells and glia share a common precursor
(Dutton et al., 2001;
Dupin and Le Douarin, 2003;
Dupin et al., 2003), both can
be generated by adult neural crest-derived stem cells
(Sieber-Blum et al., 2004;
Amoh et al., 2005;
Wong et al., 2006), and both
are affected by the erbb3b mutation and by ErbB inhibitor treatments.
ErbB signals also may expand a precursor population. If so, then incomplete
regulation could explain why the early larval pigment pattern is normal in
picasso mutants: if precursors are allocated to fill a defined number
of `embryonic/early larval niches' before filling `metamorphic niches', a
depleted total number of cells could leave metamorphic niches vacant.

In a second model, ErbB signals promote adult pigment pattern formation
non-autonomously to the metamorphic melanophore lineage. This could occur if
ErbB-expressing cells provide trophic support to metamorphic melanophore
precursors or contribute otherwise to a micro-environment where these
precursors reside. Such interactions would be analogous to the non-autonomous
mechanisms by which ErbB signals in glia promote neuronal survival and nerve
integrity (Riethmacher et al.,
1997; Chen et al.,
2003; Sharghi-Namini et al.,
2006). These observations also raise the possibility that
peripheral nerves or ganglia serve as niches for metamorphic melanophore
precursors. Consistent with this idea are the defects in ganglion development
seen here and in the accompanying study
(Honjo et al., 2008), and the
presence of cells in peripheral nerves or ganglia of other organisms that are
able to produce melanocytes and other cell neural crest derivatives
(Nichols et al., 1977;
Nichols and Weston, 1977;
Ciment et al., 1986;
Nataf and Le Douarin, 2000;
Rizvi et al., 2002;
Joseph et al., 2004).

Beyond the embryo, our data also indicate a role for ErbB signals during
metamorphosis. At this stage, ErbB signals are likely to act autonomously to
metamorphic melanophores, given their expression of erbb3b and
erbb2, but also could have non-autonomous effects if interactions
among melanophores promote the survival, proliferation or differentiation of
these cells (Parichy et al.,
2000b; Parichy and Turner,
2003b).

In conclusion, this study supports a model in which ErbB signals, which are
mediated in part through erbb3b, are required in the embryo to
establish latent precursors that will subsequently generate metamorphic
melanophores. Later, during metamorphosis, ErbB signals contribute to
melanophore development but are partly or entirely redundant with other
pathways. We speculate that erbb3b promotes the development of latent
precursors intrinsically, and also is required extrinsically to form a niche
where these cells reside until recruited at metamorphosis.

Acknowledgments

Thanks to members of the Parichy laboratory for helpful discussions and for
assistance with fish rearing, to Will Talbot's laboratory for complementation
testing of picasso against erbb3b, to Melissa Harris for
comments on the manuscript, to Dave Raible's laboratory and especially Hillary
McGraw for discussions and advice, and to Judith Eisen and Yasuko Honjo for
sharing data prior to publication. Supported by NIH R01 GM62182 and NSF IOB
0541733 to D.M.P.

Parichy, D. M., Rawls, J. F., Pratt, S. J., Whitfield, T. T. and
Johnson, S. L. (1999). Zebrafish sparse corresponds
to an orthologue of c-kit and is required for the morphogenesis of a
subpopulation of melanocytes, but is not essential for hematopoiesis or
primordial germ cell development. Development126,3425
-3436.

Elisa Martí and colleagues discuss the multiple roles played by centrosomes during embryonic growth of the vertebrate central nervous system, highlighting the links between centrosome dysfunction and microcephaly.

In September, Development held the third of its highly successful series of meetings focusing on human developmental biology. The Node ran a competition to find a meeting reporter who would share their experiences of the meeting in exchange for free registration. Competition winner Antonio Barral Gil, a PhD student in Miguel Manzanares’ Lab at CNIC in Madrid, now recollects an enriching few days in the heart of the English countryside.

Peter Baillie-Johnson was working on the generation of elongating gastruloids to mimic the first stages of axial elongation in the mouse embryo, but was limited by aggregates adhering to the surface of culture plates. A Travelling Fellowship from Development allowed Peter to visit Matthias Lutolf in Switzerland, whose lab specialises in the use of engineered PEG hydrogels that can be produced to defined physicochemical specifications. There, Peter optimised a protocol for producing gastruloids and developed a strategy for individually embedding them in small volumes of PEG hydrogels. Read more about his story here.

Where could you research take you? Join Peter and apply for the next round of Travelling Fellowships from Development by 30 November 2018.

We are pleased to announce a Workshop, organised by Benoit Bruneau and Joanna Wysocka, focusing on the field of chromatin biology and its potential for revealing exciting new insights into developmental processes. The Workshop will be held in April 2019 at Wiston House, UK. Ten funded early-career places are available - apply here before 21 December 2018.

Natalie Dyehighlights a recent preprint from Yu Sun and colleagues, describing how a new way of measuring the mechanical properties of bulk tissues in vivo identifies a stiffness gradient in the developing limb that correlates with patterns of cell migration.

Catch up with recent preLights selected for the developmental biology community in our latest Table of Contents.